Mito-VPS18 and mito-VPS39 are not able to recruit VPS41. (A) Confocal images of HeLa^VPS41KO cells transfected with VPS41^WT-V5, mito-VPS41^WT-V5, or the truncation mutant mito-VPS41^R662*-V5 (which lacks the RING domain) and labeled for V5 and endogenous TOM20. Both mito-VPS41 constructs were overlapping with TOM20-positive mitochondria. Scale bars: 20 μm. (B) Pearson’s correlation coefficient of anti-V5 (VPS41^WT-V5 and mito-VPS41^WT-V5) versus mitochondrial Tom20 as shown in A. Data show the mean ± SEM individual cell experimental replicate means, respectively. n ≥33. Statistical analysis was obtained using an unpaired t test, ****P < 0.0001. (C) HeLa^VPS41KO cells transfected with mito-VPS41^WT-V5 and immunolabeled for endogenous VPS18 and V5 as described in the figure. Scale bars: 10 µm. (D) Confocal images of HeLa^VPS18KO cells co-expressing mito-VPS18-GFP and VPS41^WT-V5 or empty vector V5 and immunostained for a V5 tag. Scale bars: 20 μm and 5 μm (inset). (E) HeLa^VPS39KO cells co-expressing mito-VPS39-GFP and VPS41^WT-V5 or empty vector V5 and immunostained for a V5 tag, showing that mito-VPS39-GFP is not able to recruit VPS41^WT-V5. Scale bars: 20 and 5 μm (inset) (F) Quantification of VPS41 ^WT-V5 and empty vector V5 recruitment to mito-VPS18-GFP as shown in D. Mito-VPS18-GFP is not able to recruit VPS41. Quantification is expressed in fold change with value 1 to the negative control V5. Statistical significance was calculated using an unpaired t test, ns: not significant. n > 28. Data show the mean ± SEM and experimental replicate means, respectively. (G) Scatter plot quantification of VPS11-HA recruitment to mito-VPS18-GFP or negative control mito-GFP as shown in J. Mito-VPS18-GFP recruits VPS11. Quantification is expressed in fold change with value 1 to VPS11-HA signal on the mask of the negative control mito-GFP. Statistical significance was calculated using an unpaired t test, ****P < 0.0001. n > 30. Data show the mean ± SEM. (H) Quantification of VPS41 ^WT-V5 and empty vector V5 recruitment to mito-VPS39-GFP as shown in E. Mito-VPS39-GFP is not able to recruit VPS41^WT-V5. Quantification is expressed in fold change with value 1 given to the mitochondrial localization of the negative control empty vector V5. Statistical significance was calculated using an unpaired t test, ns: not significant. n > 30. Data show the mean ± SEM. (I) Scatter plot quantification of VPS11-HA recruitment to mito-VPS39-GFP or negative control mito-GFP as shown in K. Mito-VPS39-GFP is able to recruit VPS11. Quantification is expressed in fold change with value 1 to VPS11-HA signal on the mask of the negative control mito-GFP. Statistical significance was calculated using an unpaired t test, ***P < 0.001. n > 30. Data show the mean ± SEM. (J) Confocal images of HeLa^VPS18KO expressing VPS11-HA and either mito-VPS18-GFP or negative control mito-GFP as indicated in the figure. Cells were immunolabeled for an HA tag. Scale bars: 20 and 5 μm (inset). (K) Confocal images of HeLa^VPS39KO expressing VPS11-HA and either mito-VPS39-GFP or negative control mito-GFP. Immunostaining for an HA tag was performed. Scale bars: 20 and 5 μm (inset).